Particles containing coated living micro-organisms, and method for producing same

ABSTRACT

Particles containing dehydrated living micro-organisms are coated with a homogeneous layer of hydrophobic substance selected to reduce the risks of degradation of the micro-organisms by physico-chemical stresses such as heat, humidity, gastric acid, or compression. A method for producing such particles includes injecting a melting hydrophobic substance into the mass of the dehydrated micro-organisms placed in a chamber swept by an air stream at controlled temperature, where the base is rotating.

The present invention relates to particles containing dehydrated livingmicroorganisms, coated with a substance which makes it possible toincrease the resistance of said microorganisms to physico-chemicalstresses such as heat, humidity, acidity or compression. The presentinvention relates equally to a method of producing such particles and tothe particles capable of being obtained by said method, and to theirapplication in pharmaceutical, dietary or food compositions.

The use of dehydrated living microorganisms in pharmaceutical, dietaryor food-processing applications, often means that during theirmanufacture, their storage or their use, these microorganisms aresubmitted to physico-chemical stresses such as for example conditions ofraised pressure, temperature, humidity or acidity. This affects theircapacity for resuming normal activity after rehydration, when theirsurvival rate is not simply extremely reduced by the aggressiveness ofsuch treatment.

Various solutions have been proposed which aim at covering themicroorganisms with a protective substance.

For example the patent U.S. 99/06746 teaches the coating of bacteriawith polymers, such as polyacrylamides, or copolymers associated withphospholipids. However, such polymers are not authorised inpharmaceutical or food products.

The patent FR 96 06215 describes the coating of bacteria with polymersor water-soluble polysaccharides, which excludes any stabilisation in anaqueous medium. In particular, for the preservation of productscontaining dehydrated microorganisms, it is imperative to guarantee avery low humidity level so that it is not possible for there to be anyresumption of activity before it is used in the desired medium.

This is why other methods have been proposed using as a protectivematerial substances which are edible and resistant to humidity.

According to the method described in the document DE 3738599, yeasts areencapsulated in fats, such as esters of fatty acids, the softeningtemperature of which is greater than 29° C. In this way the start of thepanary fermentation in fresh or frozen lumps of dough is delayed forseveral hours, exploiting the progressive softening of the coatingsubstance used. Such particles do not therefore make it possible toensure mechanical protection against compression, nor efficient thermalprotection.

The patent WO 9212234 relates to bacteria encapsulated in fatty acids,and more particularly to Enterococcus faceium encapsulated in stearicacid. The capsules are formed from a paste made up of bacteria and fattyacids closely mixed together, and are presented as microspheres ofbinding material in which the bacteria are dispersed in a random manner.Such capsules do not include a protective layer deposited regularly onthe microorganism providing chemical stability and homogenous mechanicaland thermal resistance. The method of manufacture mixing the moltenbinding material and the bacteria limits this technique to the coatingof rare microorganisms surviving treatment at high temperatures, themelting temperature of stearic acid being 70±1° C. Moreover, the size ofthe microspheres obtained is between 75 and 300 μm, which further limitsthe application of the method to the coating of initial cell clusterswhich are of a size smaller than these values (and poses furthermoreproblems of clogging during handling).

The patent U.S. Pat. No. 4,888,171 describes a granular product made upof a core, formed from crystallised sugar for example, the latter beingcoated with a composition adhering to it, formed from the mixture of abinding material, with a melting point of between 25 and 60° C., anddried bacterial cells. The product obtained has a stratified structuresurrounding the core. This product is prepared by a method according towhich the binding material is vaporised in a granulation chamber in amanner concomitant with the feeding of dried bacterial cells, theambient temperature of the chamber being between 25 and 55° C. Thus fora coating material which has a melting point greater than 60° C., thegranulation chamber will have to be kept at a temperature greater than55° C., at which the microorganisms are partially killed or are sodamaged that they then perish. This is the reason why this method isrestricted to the use of coating materials, the melting point of whichis lower than 60° C.

All the techniques previously described involve disadvantages connectedwith the necessity of reconciling aggressive manufacturing methods andfragile biological entities, which constitutes an obstacle to obtainingefficient and durable protection of living microorganisms, which has notbeen overcome up to present.

Thus the technical solutions proposed do not permit simultaneously:

-   -   preparing protective particles of living microorganisms,    -   and preserving the microorganisms during the manufacture of said        particles.

Indeed up to present, either the coating material has quite a lowmelting point so as not to damage the microorganisms during thepreparation of the particles, but it is not solid at ambient temperatureand such particles cannot ensure efficient and durable protection; orraised temperature conditions are applied to obtain the melting of thematerial which has to cover the cells, making it possible to obtainsolid particles which are resistant to ambient temperature, but whichseriously damage the viability of the microorganisms.

It appears therefore that up to present, it has not been possible toproduce, from clusters of cells of pre-existing dehydrated livingmicroorganisms, coated particles which present a high resistance tophysico-chemical stresses such as heat, humidity, gastric acidity orpressure in an efficient and durable manner, and which can be used inthe applications of dietetics, pharmacology and the food-processingindustry.

The particles according to the invention have the advantage of goodresistance to mechanical stress, excellent chemical stability in hardlyrestrictive storage conditions and for the use sought, allied with aperfect viability thanks to the perfecting of a simple process ofgeneral importance making it henceforth possible to use a large numberof substances for the coating and protection of the most varied livingmicroorganisms.

The principle of the preparation method used in the present inventionrests on the rapid cooling of micro-drops of the coating substance whenthey are injected into a granulation chamber, the chamber containing themicroorganisms to be coated being kept at a regulated temperaturecompatible with the survival of the microorganisms. The choice of thecoating substance is therefore no longer dependent on the compatibilitybetween its melting point and the temperature tolerated by themicroorganism during preparation but solely on desired criteria for thefinal product.

It is possible, for example, to use a coating material, the meltingpoint of which is greater than the temperature of the human body, suchthat the particles do not melt and remain inactive when ingested so asto release the useful microorganism or microorganisms only once they arein the stomach. If, furthermore, the selected coating material iscapable of resisting, at least partially, the attack of the gastricjuices, it will be possible to provide the useful flora beyond thegastric barrier.

The properties of high mechanical resistance are obtained by thecombination of two factors essentially. On the one hand, the coatingsubstance is chosen to be in a solid state at the preservationtemperature, and on the other hand, the particles are formed from asingle layer of hydrophobic material, covering in a homogenous fashionthe cluster of dried cells, not having any cavities showing on thesurface so as not to facilitate the formation of fissures and chemicalattacks, nor breaking and crushing.

Finally, the particles which are the object of the present invention areparticularly easy to use because they have a granulometry which providesthem with quite considerable flow properties during the preparation andhandling of the particles, the latter maintaining a fluidity comparableto that of a liquid and not causing any clogging.

Thus the present invention, by a judicious choice of different chemicaland physical parameters which will be explained hereinafter, proposesparticles provided with original properties containing livingmicroorganisms of a varied nature, and a method of producing saidparticles making it possible to reconcile the various technicalexigencies during their manufacture, preservation and use.

The solution to the problem posed resides in the realisation ofparticles made up of a cluster of dehydrated living microorganisms and alayer of homogenous hydrophobic coating.

The hydrophobic layer is composed principally of a substance selectedfrom fats, and in particular from fatty acids or waxes, either pure ormixed together. The essential characteristic of the coating layer is itsmelting point which can be between 20 and 100° C., preferably between 30and 80° C. Saturated fatty acids are preferred and in particular stearicacid. Palmitic acid can also be used for its technical qualities. Animalor vegetable waxes such as Carnauba wax are equally suitable.

To this coating layer it is possible to add other additive molecules,such as antioxidants, sugars or proteins, known to improve the stabilityof microorganisms in the conditions of manufacturing and/or preservingmicroorganisms coated according to the invention.

The coating layer covers the cell clusters in a homogenous manner, i.e.its thickness varies very little and above all it has a uniformstructure and does not contain inclusions or cavities which could affectthe regularity of its surface, and consequently its rigidity and itscohesion. It is also chemically homogenous.

The choice of compounds compatible with the expected uses of theparticles according to the invention is of course recommended. Inparticular one will chose by preference materials which meet thecriterion of food quality.

The microorganisms intended to be coated are living cells which havebeen dried by lyophilisation, atomisation or on a fluidised bed, so thatthey can be revived. These techniques are well known to the personskilled in the art. After crushing, cell clusters are obtained which arein the form of a more or less fine powder. These cell clusters can besubstantially spherical, ovoid or elongated, smooth or rough, regular orirregular. Their size is between several microns and severalmillimetres.

A great variety of microorganisms, even the most fragile and the mostsensitive to the conditions of the medium, are capable of entering intothe preparation of particles according to the invention, insofar as themethod of manufacturing the particles is not at all aggressive incomparison with techniques known previously. Let us quote the bacteriafor food, dietary or pharmaceutical use such as for example thelactobacilli, especially Lactobacillus casei, L. casei rhamnosus, L.acidophilus, L. bulgaricus, L. brevis, L. helveticus, thebifidobacteria; streptococci, especially Streptococcus thermophilus,Streptococcus salivarius; Lactococci such as Lactococcus lactis; thePediococci especially Pediococcus acidilactici; the enterococci; theyeasts of the genus Saccharomyces (in particular Saccharomycescerevisiae, S. boulardii), Kluyveromyces, etc. The choice of one or amixture of these organisms will be determined above all by the finalapplication for which it is intended, the relatively gentlemanufacturing conditions proposed by the present invention notconstituting the limiting criterion.

The dry powders of the above-mentioned microorganisms, obtained bylyophilisation, by atomisation or by drying in a fluidised bed, aresuitable as the starting material, this list not being restrictive.Thus, the particles according to the invention can also containmicroorganisms belonging to other species and being of interest forother fields of application, such as cosmetics, the protection of theenvironment, industrial processes requiring for example the regulatedsupply of additives, or any other field.

In the particles according to the invention, the proportion of coatingmaterial in relation to the quantity of microorganisms is between 10 and99% by weight, advantageously between 30 and 80%. If the coating layeris too fine, the protection will not be sufficient; conversely, if thelayer is too thick, the release of the microorganisms will be longer.This parameter will thus be adjusted according to the intended finalapplication, depending on the desired speed of release of themicroorganisms.

The concentration of viable bacteria in the coated particles is inducedfrom the relative proportion of the coating substance and the driedpowder used. Indeed, the number of microorganisms per gram of driedpowder being known before coating, it is possible to calculate theconcentration per particle after coating. This theoretical value iscompared with the actual value measured by standard methods, as acontrol.

The concentration of viable bacteria is expressed as UFC/g: unit formingcolony per gram of coated particles of the relevant microorganism. Themeasuring method is based on microbiological counting on a Petri dish,after appropriate dilution, according to the techniques known to theperson skilled in the art.

The final particles have a controlled size, depending on the size of theclusters of microorganisms of the starting powder and on the thicknessof the deposited coating, with an average diameter which can varybetween 100 and 5000 μm, preferably between 300 and 2000 μm. It ispossible to vary the size of the freeze-dried clusters depending on theintended application, using any technique at the disposition of theexpert, for example by prolonged grinding.

The present invention relates also to a method of producing particles ofcoated microorganisms such as described previously, consisting ininjecting into a chamber molten hydrophobic material, in a mass ofmicroorganisms permanently crushed by rotation of the disc which acts asthe base of the chamber, and swept by a current of dry air at a fixedtemperature.

It has in fact been found in a surprising manner that it was possible toobtain, by injecting molten hydrophobic products into a chamber wherethe particles were subjected to rotary agitation and to sweeping by anairflow, the parameters being judiciously selected, coated particleswhich have the desired characteristics of physical and chemicalstability.

The coating method uses a standard commercial apparatus, called agranulator, available in different sizes compatible with productionvolumes from several hundreds of grams to several hundreds of kilogramsper operation.

FIG. 1 gives a schematic representation of the device. Referring to thisdrawing, the device comprises a stainless steel chamber, the base (2) ofwhich is made up of a disc which is animated with a rotary movement by amotor (3). An airflow is injected via the space (4) between the base (2)and the body (1) of the chamber. The air escapes from the chamber via afilter (5) placed in the upper portion of the chamber. The mass ofpowdered microorganisms (6) is agitated by the rotation of the disc (2).A nozzle (7) permits the injection, by means of a pump (8), of thecoating product kept at a temperature above its melting point in atemperature-controlled receptacle (9).

In the implementation of the method according to the invention, theparameters of which the choice is critical for obtaining a homogenouscoating are the temperature of the coating product, the temperature ofthe air sweeping the chamber, the speed of rotation, the rate ofinjection of the coating, and the mass of products used. Examples 1 to 4illustrate particular embodiments of the invention without limiting thescope however.

The temperature of the coating substance placed in thetemperature-controlled receptacle (9) can be between 30 and 120° C.,preferably between 60 and 120° C. In any case, it ought to be greaterthan the melting point of said substance, whether this is a pure productor a mixture.

The temperature of the air sweeping the granulation chamber is between10 and 50° C. It is strictly controlled, so that at the moment ofinjecting the molten coating, the rise in temperature experienced by themicroorganisms does not exceed a few degrees, at maximum 5° C. Forcertain microorganisms which cannot tolerate temperatures greater than40° C. the temperature of the chamber will obviously be reduced.

The speed of rotation and the rate of injecting the coating areinterdependent parameters and connected to the masses of the productsused. The speed of rotation is generally between 50 and 500 rpm(rotation per minute). The injection of the coating can be carried outwith the aid of one or more nozzles distributed over the periphery ofthe chamber.

All the parameters are also adjusted as a function of the shape of theinitial cell clusters, and of the nature of the coating product.

The advantages of the method such as described above lie in thepossibility of coating in a non-aggressive manner microorganism cells ofvaried shape and size, with a resistant hydrophobic layer permittingefficient and durable protection, as well as in the numerouspossibilities of application offered by such properties because of thegreat diversity of the organisms which can be coated in this way,especially in the pharmaceutical, dietary or food fields.

The object of the invention is also the use of the previously describedparticles in the pharmaceutical, dietary or food-processing fields. Inparticular the invention permits the addition of microorganisms todifferent food products such as cereals, confectionery, powdered milk,in tablets etc. . . . whilst permitting a sufficient viability of saidmicroorganisms during the manufacture of said products and for theduration of the storage preceding consumption. It also makes it possibleto protect the microorganisms against gastric acidity for betteractivity in the intestine. Because of the original properties of theparticles according to the invention, numerous other uses can also beenvisaged.

The following examples illustrate in a non-restrictive manner,embodiments of the present invention and the parameters used for theproduction of different types of particles.

EXAMPLE 1

Coating of Lactobacillus acidophilus with Stearic Acid

600 g of a freeze-dried powder of bacteria Lactobacillus acidophilus,strain R052, registered at the CNCM under no. 1-1722, marketed by theInstitut Rosell, 8480 boulevard Saint Laurent, Montreal, Canada areintroduced into a granulator with a flow of air, brand name Glatt, modelGPCG1, in a “rotor” configuration with a capacity of 3 litres. 900 g ofstearic acid (Fluka, reference 85683, melting point mp=69–71° C.) areintroduced into the temperature-controlled receptacle (9).

The coating operation is conducted according to the followingparameters:

-   -   rotor speed: 300 rpm    -   speed of the air in the chamber: 3 to 4 m/s or 40% opening of        the entry flap    -   pulverisation pressure of the coating material: 2 bars    -   coating rate: 40 g/minute    -   temperature of the pulverisation air: 120° C.    -   temperature of the stearic acid: 100° C.    -   temperature of the incoming air: 30° C.    -   temperature of the product: 32–35° C.

At the end of the operation, the granulator is empty and the particlesare collected and stored in airtight sachets.

The particles thus obtained have the following characteristics:

-   The average diameter of the particles is 400 μm, with 92% between    100 and 600 μm.-   The content of coating material is 60%.-   The concentration of viable bacteria of the starting powder is    3,2.10¹¹ UFC/g (units forming colonies per gram), that of the coated    particles is 1,2.10¹¹ UFC/g of powder introduced initially.

EXAMPLE 2

Coating of Lactobacillus casei by a Mixture of Fatty Acids

600 g of a freeze-dried powder of Lactobacillus casei, strain EQ 85(registered at the CNCM under the number MA 64U), marketed by LallemandSA, 15130 Saint Simon (in Canada?), are introduced into the samegranulator as the one used in example 1 and the coating is placed in thetemperature-controlled receptacle. The coating product is a mixture ofstearic acid and palmitic acid in equal parts, the melting point ofwhich is mp=55° C., marketed by Exaflor (47, allée de Chanteraine, 91190Gif sur Yvette, France), under the designation Stéarine™ 50/50.

The parameters used are the same as those mentioned in example 1, withthe exception of:

-   -   the temperature of the coating: 80° C.    -   the temperature of the incoming air: 25° C.    -   the temperature of the product: 28–32° C.

The particles obtained have the following characteristics:

-   They have an average diameter of 750 μm with 90% between 100 and    1000 μm.-   The content of fatty acid is 60%.-   The concentration of viable bacteria of the initial powder is    4,8.10¹¹ UFC/g, that of the coated particles is 1,6.10¹¹ UFC/g.

EXAMPLE 3

Coating of Saccharomyces cerevisiae by a Vegetable Wax

750 g of a preparation of dry yeast Saccharomyces cerevisiae, straindeposited at the CNCM under the number I 1079, marketed under the brandname Levucell SB, by Lallemand Sarl 15130 Saint Simon, Canada areintroduced into the granulator with an airflow used in example 1, andcoated with 750 g of a vegetable wax, Carnauba wax, the melting point ofwhich is mp=83–88° C. (marketed by Exaflor, Gif sur Yvette, France).

The parameters are identical to those of example 1, apart from:

-   -   the temperature of the wax: 120° C.    -   the temperature of the incoming air: 40° C.    -   the temperature of the product: 45–48° C.

The particles obtained have the following characteristics:

-   They have an average diameter of 1200 μm, with 90% between 500 and    2500 μm.-   The content of fatty acid is 50%.    -   The concentration of viable cells of the initial powder is        3.10¹⁰ UFC/g, that of the coated particles is 1,45.10¹⁰ UFC/g of        the starting powder.

EXAMPLE 4 Coating of Pediococcus acidilactici in a Mixture of FattyAcids

80 kg freeze-dried powder of Pediococcus acidilactici, strain filed theCNCM under the number MA 18/5M, marketed by Lallemand SA, 15130, SaintSimon, Canada under the brand name Bactocell, are introduced into aGLATT™ granulator, model CRG200, with a capacity of 450 litres, equippedwith two injection nozzles. The freeze-dried cells are coated with 160kg of Stéarine 50/50(TM) (Exaflor, Gif sur Yvette, France).

The parameters for preparing particles are:

-   -   speed of the rotor: 120 rpm    -   air flow: 1500 to 2000 m³/h    -   pulverisation pressure: 5 bars    -   flow of the fatty acid: 800 g/mn (400 g/mn per nozzle, on two        nozzles)    -   temperature of the coating material: 80° C.    -   temperature of the pulverisation air: 120° C.    -   temperature of the incoming air: 25° C.    -   temperature of the product at 30° C.±2° C.

The particles obtained have the following characteristics:

-   The average diameter is 400 μm, with 87% between 100 and 600 μm.-   The content of coating material is 75%.-   The concentration of viable bacteria of the initial powder is 3.10¹¹    UFC/g, that of the coated particles 7,5.10¹⁰ UFC/g.

EXAMPLE 5

Thermal Stability

The viability of the bacteria Lactobacillus acidophilus in thefreeze-dried powder used in example 1 on the one hand, and in the coatedparticles obtained according to this same example 1 on the other hand,have been studied in the following conditions:

Samples of 10 grams of each preparation are introduced into sealedtubes, kept in a water bath at 50° C. A tube of each preparation iswithdrawn from the water bath after 1 hour, 4 hours, 7 hours and 24hours and the concentration of viable bacteria is immediatelydetermined.

The results are listed in table 1 below. The concentrations areexpressed in UFC/g and in a percentage of the concentration of eachsample at T=0:

TABLE 1 1 2 3 4 Time 0 month months months months Freeze- 3,1.10⁹1,3.10⁹ 1,2.10⁹ 9,9.10⁸ 4,8.10⁸ dried (100%) (42%) (38%) (32%) (15%)powder Coated 7,5.10⁸ 6,4.10⁸ 5,5.10⁸ 5,3.10⁸ 5,2.10⁸ particles (100%)(85%) (73%) (71%) (69%)

The stability at 50° C. of the bacteria in the form of particles coatedaccording to the present invention is clearly improved by comparisonwith that of the bacteria in the form of freeze-dried powder.

EXAMPLE 6

Stability in Powdered Milk

The viability of the bacteria Pediococcus acidilactici in thefreeze-dried powder used in example 4 on the one hand, and in the coatedpowders obtained according to this same example 4 on the other hand, hasbeen studied in the following conditions:

The preparations of bacteria are mixed into powdered milk (brand nameRégiliat) at a rate of 1% by weight of bacterial preparation for 99% ofmilk powder. The mixtures are divided into samples of 100 grams inwelded polythene sachets. The sachets are kept at 30° C. in anincubator. Each month, a sachet of each mixture is analysed for itscontent of viable bacterial.

The results are listed in table 2 below, the concentrations areexpressed in UFC/g and in a percentage of the concentration of eachsample at T=0.

TABLE 2 Time 0 1 h 4 h 7 h 24 h Freeze- 3,1.10¹¹ 2,9.10¹¹ 1,1.10¹¹9.10¹⁰ 2,5.10⁹ dried (100%) (93%)   (35%)   (29%) (8%) powder Coated1,2.10¹¹ 1,2.10¹¹ 1,2.10¹¹ 1,1.10¹¹ 9.10¹⁰ particles (100%) (100%)(100%) (92%) (69%)   

In the presence of powdered milk at 30° C., the stability of thebacteria in the form of particles coated according to the presentinvention is clearly improved by comparison with that of the bacteria inthe form of freeze-dried powder.

EXAMPLE 7

Gastric Stability

The stability of the bacteria Lactobacillus acidophilus in conditionssimulating passing into the stomach have been studied according to thefollowing protocol:

Two samples are prepared, one from the freeze-dried powder such as usedin example 1 and the other from particles coated according to this sameexample 1. For each sample, 1 gram of freeze-dried powder or of coatedparticles is introduced into a flask containing 100 ml of a hydrochloricacid solution 0.1N (pH 1.2). The flask is placed under agitation in awater bath at 37° C. for one hour. The suspension is then centrifuged,and the remainder is taken up into 100 ml of phosphate buffer with a pHof 7.0. The residual concentration of viable bacteria is then determinedand compared with that of a control in which the hydrochloric acid isreplaced by a buffered solution with a pH of 7.0.

In these conditions, the Lactobacillus acidophilus bacteria in the formof freeze-dried powder are practically entirely destroyed (survival rateless than 0.01%) whilst the same bacteria contained in the coatedparticles have a survival rate of 15%.

The stability of the bacteria Lactobacillus casei has been studiedaccording to the same protocol. The Lactobacillus casei bacteriacontained in the freeze-dried powder such as used in example 2 arepractically entirely destroyed by the gastric test (survival rate lessthan 0.01%) whilst the same bacteria contained in the particles coatedaccording to this same example 2 have a survival rate in this same testof 25%.

EXAMPLE 8

Stability Against Compression

Tablets based on the microorganisms Lactobacillus acidophilus,Lactobacillus casei, Saccharomyces cerevisiae and Pediococcusacidilactici, dried by lyophilisation, such as described in examples 1,2, 3 and 4 respectively, are prepared according to the followingprotocol:

For each microbial sample, the freeze-dried powder is mixed at a rate of5% by weight into an excipient composed in the following manner:

-   49% sorbitol, (reference Neosorb 60w UPSA),-   49% lactose, (Fast flow Seppic)-   2% magnesium stearate (UPSA)

Each mixture is introduced successively into a tabletting apparatus ofan alternating type, model EKOD marketed by Korsch. The compressionforces exerted are 17500 N on the upper piston and 16400 N on the lowerpiston.

The concentrations of viable cells are determined in each mixture beforecompression, then in the tablets. The results, expressed as a percentageof survival after compression, are listed in the following table 3:

TABLE 3 Lacto- Lacto- bacillus bacillus Saccharomyces PediococcusSpecies acidophilus casei cerevisiae acidilactici Non-coated 44% 3% 2%48% powder Coated 98% 10% 16% 100% particles

Survival after compression of the bacteria or yeasts contained in thecoated particles is clearly improved by comparison with thecorresponding non-coated powders.

1. A method of coating dehydrated living microorganisms characterised inthat a molten hydrophobic substance having a melting point higher than60° C. is injected at a temperature of at least 5° C. higher than themelting temperature of said hydrophobic substance, said injectiontemperature being between 65° C. and 120° C., into a chamber containingsaid microorganisms which are agitated by rotation of the base of saidchamber and swept by a flow of air at a temperature of between 10° C.and 50° C., said airflow having a sweeping speed so that the temperaturein the chamber does not exceed by more than 5° C. the viabilitytemperature of said microorganisms, and wherein particles made of acluster of dehydrated living microorganisms and a homogenous hydrophobiccoating layer are obtained.
 2. The method of coating dehydrated livingmicroorganisms according to claim 1, characterised in that the speed ofrotation of the base of said chamber is between 50 and 500 rpm.
 3. Themethod of coating dehydrated living microorganisms according to claim 1,characterised in that said hydrophobic coating substance has a meltingpoint of between 60° C. and 100° C.
 4. The coating method according toclaim 1, characterised in that said hydrophobic coating substance isselected from the group consisting of fats, fatty acids, waxes, andmixtures thereof.
 5. The coating method according to claim 1,characterised in that said dehydrated living microorganisms are selectedfrom the group consisting of lactobacilli, bifidobacteria, streptococci,streptococci faecalis, pediococci, yeasts, and a mixture thereof.
 6. Thecoating method according to claim 1, characterised in that theproportion of said injected coating substance is between 10 and 99%, byweight of the final particles.
 7. The method according to claim 1,wherein the chamber temperature is between approximately 30 and 55° C.8. The method according to claim 4, wherein the melting point is between60° C. and 80° C.
 9. The method according to claim 6, wherein theproportion of said injected coated substance is between 30 and 80%. 10.A method of coating dehydrated living microorganisms, comprising:introducing said microorganisms into a chamber having a base; agitatingsaid microorganisms by rotation of the base of said chamber; providing aflow of air at a temperature of between 10° C. and 50° C., said airflowhaving a sweeping speed so that the temperature in the chamber does notreach a temperature of more than 5° C. of the viability temperature ofsaid microorganisms; and thereafter injecting a molten hydrophobicsubstance having a melting point higher than 60° C. into said chambercontaining said microorganisms at a temperature of at least 5° C. higherthan the melting temperature of said hydrophobic substance, saidinjection temperature being between 65° C. and 120° C., and whereinparticles comprising dehydrated living microorganisms having ahomogenous hydrophobic coating layer covering said microorganisms areobtained.
 11. The method of coating dehydrated living microorganismsaccording to claim 10, wherein the speed of rotation of the base of saidchamber is between 50 and 500 rpm.
 12. The method of coating dehydratedliving microorganisms according to claim 10, wherein said hydrophobiccoating substance has a melting point of between 60° C. and 100° C. 13.The coating method according to claim 10, wherein said hydrophobiccoating substance is selected from the group consisting of fats, fattyacids, waxes, and mixtures thereof.
 14. The coating method according toclaim 10, wherein said dehydrated living microorganisms are selectedfrom the group consisting of lactobacilli, bifidobacteria, streptococci,streptococci faecalis, pediococci, yeasts, and a mixture thereof. 15.The coating method according to claim 10, wherein the proportion of saidinjected coating substance is between 10 and 99%, by weight of the finalparticles.
 16. The method according to claim 10, wherein the chambertemperature is between approximately 30 and 55° C.
 17. The methodaccording to claim 13, wherein the melting point is between 60° C. and80° C.
 18. A method of coating dehydrated living microorganisms,comprising: introducing a cluster of cells comprising saidmicroorganisms into a chamber having a base; agitating said cluster ofcells comprising said microorganisms by rotation of the base of saidchamber; providing a flow of air at a temperature of between 10 and 50°C. into said chamber, said airflow having a sweeping speed so that thetemperature in the chamber does not exceed by more than 5° C. of theviability temperature of said microorganisms; and thereafter providing amolten hydrophobic substance having a melting point higher than 60° C.into said chamber containing said cluster of cells comprising saidmicroorganisms at a temperature of at least 5° C. higher than themelting temperature of said hydrophobic substance to obtain particlescomprising said cluster of cells comprising said microorganisms and ahomogenous hydrophobic coating layer.
 19. The method according to claim18, wherein the melting point is between 60° C. and 100° C.
 20. Themethod according to claim 18, wherein the melting point is between 69°C. and 100° C. and the injection temperature is between 74° C. and 120°C.